Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide, with roughly 500,000 new cases and 300,000 cancer-related deaths globally occurring each year. Our group has comprehensively characterized the genomic alterations in HNSCC, and we previously reported that NOTCH1 is among the top genes mutated in this disease. The spectrum of NOTCH1 mutations we found suggested the gene is a tumor suppressor in HNSCC, and we recently published experiments which confirmed this by demonstrating restoration of the NOTCH1 gene to HNSCC cell lines with naturally occurring NOTCH1 mutations impairs their growth both in vitro and in vivo. We now have data demonstrating that NOTCH signaling similarly impairs the growth of HNSCC cell lines lacking mutation and expressing wild type NOTCH1 receptors, raising the possibility that this pathway may someday be exploited in a targeted fashion to treat patients with HNSCC. Our preliminary data also show that NOTCH activation in HNSCC lines inhibits cell migration, activates a program of changes in molecules that regulate cell adhesion and adhesion-dependent signaling, and induces genes associated with differentiation and tumor suppression. In addition, we have novel data linking NOTCH reactivation to suppression of proto-oncogenes, a-Catulin and Axl kinase, which have been shown to regulate malignant properties of tumors - suggesting the possibility that loss of these proteins could mediate the tumor suppressive phenotypes in HNSCC induced by NOTCH activation. These observations support a central hypothesis that NOTCH inactivation results in more aggressive tumors because NOTCH activation disrupts adhesion-dependent signaling, causing cells to become more differentiated, lose proliferative capacity, become less migratory or invasive, and upregulate tumor suppressor genes. To test this hypothesis, the cellular and molecular mechanisms by which NOTCH activation inhibits malignant properties will be determined in HNSCC cell lines harboring mutant or wild type NOTCH1 receptors, while the impact of NOTCH mutation on tumor aggressiveness will be studied in genetically-engineered mice and cell lines, and in a clinical patient cohort. Th proposed work should advance our knowledge regarding the biological and clinical significance of the NOTCH pathway in HNSCC, which is critical in order to correctly understand how the pathway may be used for therapy or prognosis in this disease. As inactivating NOTCH1 mutations have now been detected in squamous cell carcinomas from lung and esophagus, knowledge gained from this work could have far reaching implications in our understanding and treatment of solid tumors from multiple types of cancer.